The long term goal of the proposed studies is to develop a detailed understanding of the mechanism by which eukaryotes replicate their DNA. DNA replication is an extremely complicated and poorly understood process that involves a host of DNA polymerases and accessory proteins. In order to better understand replication, the primase-catalyzed synthesis of an RNA primer and elongation of that primer by DNA polymerase alpha will be examined. The specific aims of this proposal are: 1. Identify interactions between pol alpha and a dNTP that result in high fidelity and efficient polymerization of correct dNTPs. Protein mutagenesis in conjunction with substrate analogs will be used to elucidate these interactions. 2. Determine how pol alpha integrates the differences between right and wrong dNTPs into its catalytic cycle in order to accurately replicate DNA. Pre-steady-state kinetic approaches will be used to determine how pol alpha discriminates against incorrect, natural dNTPs and how the chemical differences between right and wrong dNTPs are integrated into the catalytic cycle. 3. Develop a thorough understanding of NTP recognition by primase and generate novel base-pairs that primase efficiently replicates. New base-pairs will be generated based on how primase chooses whether or not to polymerize a NTP. 4. Determine how pol alpha interacts with dNTPs and template lesions when elongating primase-synthesized primers, and why pol allpha elongates primase-synthesized primers quite differently than exogenously added primers. Steady-state kinetic approaches and photocrosslinking approaches will be used to understand the differences between primase synthesized and exogenously added primers. DNA replication inhibitors comprise a major class of anti-cancer and anti-viral chemotherapeutics, with approximately 2 dozen clinically useful compounds and many more in various stages of development. Thus, a better understanding of the enzymes involved in DNA replication could lead to the development of new chemotherapeutics. A key question regarding DNA and RNA polymerases is how they discriminate between correct and incorrect (d)NTPs. Since DNA polymerase-mediated mutagenesis can lead to cellular transformation, understanding how DNA polymerases discriminate between correct and incorrect dNTPs may lead to a more fundamental understanding of carcinogenesis.